Method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension reference listed drug (rld)

ABSTRACT

The invention provides methods for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD.

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention relates to methods for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD.

BACKGROUND OF THE INVENTION

A peptic ulcer is a focal mucosal defect with inflammatory cell infiltration and coagulation necrosis extending through the muscularis mucosae. The overall concept of the pathophysiology of peptic ulcer disease (PUD) is that there is a disturbed equilibrium between the aggressive and protective factors, with a very complex interplay between the factors. Peptic ulcers, characterized by a damaged gastric mucosal barrier, tend to occur within the section of the gastrointestinal (GI) tract that is in contact with gastric juice containing acid and pepsin. The gastric mucosal barrier is the feature of the stomach that allows it to safely contain the gastric acid required for digestion. The predilection sites for ulcers are the vicinity of mucosal junctions such as the transitional zone between the esophageal and gastric mucosa and the gastro-duodenal junction. PUD is commonly associated with gastritis and it is generally accepted that colonization by the bacterium H. pylori is causatively related to gastritis associated with PUD.

Carafate® suspension (Sucralfate suspension) is available in the United States as an oral suspension. It was approved in 1993 for the treatment of active duodenal ulcers. It is also widely used for the treatment of PUD, gastritis due to gastroesophageal reflux disease (GERD), irritable bowel syndrome (IBS), non-erosive reflux disorder (NERD) and functional dyspepsia. Sucralfate may also be used to prevent recurrent ulcers after healing of the ulcer has been achieved. It is also used to relieve or prevent ulcers caused by non-steroidal anti-inflammatory drugs (NSAIDs).

The mechanism of action of sucralfate is not fully understood and several possible mechanisms have been suggested. In general, it is widely believed that sucralfate exerts its anti-ulcer activity by forming an ulcer-adherent complex with the proteinaceous exudate at the ulcer site, thus covering the ulcer site and protecting it against further attack by aggressive factors such as acid, pepsin and bile salts.

A need remains for an oral Sucralfate suspension that is safe, efficacious and cost-efficient generic drug product of Sucralfate suspension that can be developed and made available to the patient population. This discovery addresses that need by identifying sucralfate suspensions that are bioequivalents of sucralfate suspension RLD.

SUMMARY OF THE INVENTION

The present invention provides, for the first time, methods for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprising contacting the sucralfate suspension sample with a bile acid, bile salt and/or conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD.

The present invention additionally comprises contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system, quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD.

The invention further provides a method for obtaining a sucralfate suspension sample that is a bioequivalent of a sucralfate suspension RLD by determining in vitro bioequivalence.

The invention also provides a method for reducing or alleviating the symptoms associated with an ulcer in a patient suffering from the ulcer comprising administering to the patient an effective amount of any one or more of the sucralfate suspension samples identified by the methods herein thereby reducing or alleviating the symptoms associated with an ulcer in the patient.

The biological outcome of the formulation depends on the similarity of the generic formulation with that of the RLD in Pharmaceutical Equivalence (PE) and Bioequivalence (BE). Taking into consideration, the complex nature of the Active Pharmaceutical Ingredient (API; i.e. Sucralfate), the locally acting nature of Sucralfate, and the multiple postulated modes of action of Sucralfate, establishing PE alone is insufficient to establish Therapeutic Equivalence (TE) of this product. Establishing BE via a clinical endpoint study as previously recommended by the USFDA has not been a successful approach to date. The current invention proposes an in-vitro based strategy that relies on orthogonal measurements of action (association with bile acids) and effect (Cytoprotection) to characterize product performance and demonstrate TE of the test product to the RLD.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Representative standard curve for TDC detection.

FIG. 2. Binding of TDC to Sucralfate at different concentrations of TDC.

FIG. 3. Dose-response for Sucralfate binding to TDC using four lots of RLD product. Data represented as mean±SD.

FIG. 4. Dose-response for TDC binding to Sucralfate using prototype Test formulation (Test_50).

FIG. 5. Dose-response for TDC binding to Sucralfate using further optimized test formulation (Test_66).

FIG. 6. Comparative performance assessment of the initial prototype test lot (Test_50) and further optimized test formulation (Test 66) for Sucralfate binding to TDC using the performance range criteria from four lots of RLD.

FIG. 7. Optimization of pH condition for cytoprotective assay. Comparison of TEER measurements between the control treatment and the indomethacin treatment at two different apical pH conditions (6.0 and 7.4). Results are presented as mean SD.

FIG. 8. Dose-response of Indomethacin. Results are presented as mean±SD.

FIG. 9. Assay duration optimization evaluation. Results are presented as mean SD.

FIG. 10. Evaluation of assay reproducibility using one lot of RLD product using Sucralfate concentration of 2.5 mg/mL. Results are presented as mean±SD.

FIG. 11. Evaluation of assay reproducibility using one lot of RLD product using Sucralfate concentration of 5.0 mg/mL. Results are presented as mean SD.

FIG. 12. Dose response of Sucralfate using one lot of RLD product (RLD_04). Data presented as mean±SD.

FIG. 13. Evaluation of inter-lot variability in cytoprotective ability using four lots of RLD product at 2.5 mg/mL Sucralfate concentration. Results presented as mean SD.

FIG. 14. Evaluation of inter-lot variability in cytoprotective ability using four lots of RLD product at 5.0 mg/mL Sucralfate concentration. Results presented as mean SD.

FIG. 15. Comparison of cytoprotective ability between the test and RLD products. Results are presented as mean SD.

FIG. 16. Comparative performance assessment of the initial prototype test lot (Test_50) and the further optimized test lot (Test_66) using the performance range criteria from four lots of RLD at 2.5 mg/mL concentration.

FIG. 17. Comparative performance assessment of the initial prototype test lot (Test_50) and the further optimized test lot (Test_66) using the performance range criteria from four lots of RLD at 5.0 mg/mL concentration.

FIG. 18. Graph of unformulated sucralfate (API), sucralfate suspension test formulations (TEST_43, TEST_75 and TEST_06) and different lots of reference listed drugs (RLD_72, RLD_66, RLD_63, RLD_23, RLD_10 and RLD_02) in maintaining percent of control TEER value for a Caco-2 cell monolayer culture in the presence of sucralfate and indomethacin. Error bars: standard error of the mean, SEM; dotted lines: range of the RLDs.

FIG. 19. Graph of cell-free apparent permeability (P_(app)) values calculated for diffusion or migration of a representative bile salt (TDC) from upper to lower chamber (compartment) of a well in a Corning Transwell® plate in the presence of a sucralfate barrier for unformulated sucralfate (API), sucralfate suspension test formulation (TEST_43, TEST_75 or TEST_06) or a different lot of reference listed drug (RLD_72, RLD_66, RLD_63, RLD_23, RLD_10 or RLD_02). Error bars: standard deviation of the mean, SD; dotted lines: upper range of the RLD_63 and lower range of the RLD_66.

FIG. 20. Graph of Langmuir binding capacity, k₂, determined for adsorption of a representative bile salt, TDC, by sucralfate in sucralfate suspension test formulations and multiple replicates of the RLD lot of the Carafate suspension product RLD_02 (error bars: standard deviation of the mean, SD; dotted lines: range of the RLD_02).

DETAILED DESCRIPTION OF THE INVENTION

All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.

Sucralfate is an hydrous basic aluminium salt of sucrose octasulfate (e.g., CAS Number: 54182-58-0; U.S. Pharmacopeial USP 35 monograph). In one embodiment, it has the chemical structure of:

In another embodiment, sucralfate has a chemical formula Al₈(OH)₁₆(C₁₂H₁₄O₃₅S)[Al(OH)₃]_(x)[H₂O]_(y) in which x=8 to 10, and y=22 to 31. Sucralfate may contain the equivalent of not less than about 30.0 percent and not more than about 38.0 percent of sucrose octasulfate (C₁₂H₁₄O₃₅S₈). Typically, between about 15.5% and about 18.5% of aluminum may be found in Sucralfate.

The term “RLD” or RLD refers to an approved drug product to which new generic versions are compared to show that they are bioequivalent. In one example, a Sucralfate Suspension RLD is Carafate® (Forest Laboratories, Inc.; Application Number: N019183; approval date: Dec. 16, 1993) which is a RLD in the United States and can be found in U.S. FDA's Orange Book.

A “suspension” is a liquid dosage form that contains solid particles dispersed in a liquid vehicle. These solid particles include drug substance, and may include excipients such as swellable polymeric muco-adhesives such as celluloses. In addition to the solid particles and vehicle, the suspension may include excipients, such as microcrystalline cellulose, colloidal silicon dixode, methyl cellulose as suspending agents, glycerin and sorbitol as bodying agents, simethicone as anti-foaming agent, methyl paraben as a preservative, food colorant and flavoring. Vehicle may be aqueous, non-aqueous or a mixture of both. In one embodiment, the vehicle is an aqueous solution or water. Merely by way of example, each 10 mL of a Sucralfate suspension may contain 1 g±10% of Sucralfate.

The term “acid” refers to a chemical compound that, when dissolved in water, gives a solution with a pH less than 7. The “acid” can be organic. It can have a pKa in the range of e.g., 2-5. Examples of acids suitable for the invention include, but are not limited to, bile acid, tartaric acid, adipic acid, succinic acid, citric acid, benzoic acid, acetic acid, ascorbic acid, edetic acid, fumaric acid, lactic acid, malic acid, oleic acid, sorbic acid, stearic acid, palmitic and boric acid or mixtures thereof.

The term “bile acid” may be any one or more of allocholic acid (3alpha,7alpha,12alpha-trihydroxy-5alpha-cholanoic acid; CAS: 2464-18-8), 5alpha-deoxycholic acid (3alpha,12alpha-dihydroxy-5alpha-cholan-24-oic acid), bitocholic acid, chenodeoxycholic acid (3alpha,7alpha-dihydroxy-5beta-cholan-24-oic acid; CAS: 474-25-9), cholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 81-25-4), deoxycholic acid (3alpha,12alpha-dihydroxy-5beta-cholanic acid; CAS: 83-44-3), glycochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid; CAS No.: 640-79-9), glycocholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oylglycine; CAS: 475-31-0), hyocholic acid (3alpha,6alpha,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 547-75-1), hyodeoxycholic acid (3α,6α-Dihydroxy-5β-cholan-24-oic acid; CAS: 83-49-8), isochenodeoxycholic acid (3beta,7alpha-dihydroxy-5beta-cholanic acid; CAS: 566-24-5), 3beta,12alpha-Dihydroxy-5beta-cholanoic acid (CAS: 570-63-8), isolithocholic acid (3beta-Hydroxy-5beta-cholan-24-oic acid; CAS: 1534-35-6), isoursodeoxycholic acid (3beta,7beta-dihydroxy-5beta-cholan-24-oic acid; CAS: 78919-26-3), 12-epideoxycholic acid, lithocholic acid (3alpha-hydroxy-5beta-cholanic acid; CAS: 434-13-9), alpha-muricholic acid (3alpha,6beta,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 2393-58-0), beta-muricholic acid (3alpha,6beta,7beta-trihydroxy-5beta-cholan-24-oic acid; CAS: 2393-59-1), omega-muricholic acid, murideoxycholic acid (3alpha,6beta-dihydroxy-5beta-cholanic acid), beta-phocaecholic acid (CAS: 105369-89-9), taurochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-11H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CAS No.: 516-35-8), taurocholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CAS No.: 81-24-3), taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7), ursocholic acid (3alpha,7beta,12alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 2955-27-3), ursodeoxycholic acid (3alpha,7beta-dihydroxy-5beta-cholan-24-oic acid; CAS: 128-13-2), and vulpecholic acid (1alpha,3alpha,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 107368-95-6).

The bile acid may be “unconjugated bile acid” such as cholic acid or chenodeoxycholic acid.

A “conjugated bile acid” may be obtained from bile acid or bile salt. For example, cholic acid or chenodeoxycholic acid may be conjugated with taurine or glycine to produce glycocholic acid or taurocholic acid in the case of cholic acid or glycochenodeoxycholic acid or taurochenodeoxycholic acid in the case of chenodeoxycholic acid.

A “conjugate base of a bile acid” is a bile acid anion. Bile acid anion may be selected from, but not limited to, allocholate, 5alpha-deoxycholate, bitocholate, chenodeoxycholate, cholate, deoxycholate, glycochenodeoxycholate, glycocholate, hyocholate, hyodeoxycholate, isochenodeoxycholate, 3beta,12alpha-Dihydroxy-5beta-cholanoate, isolithocholate, isoursodeoxycholate, 12-epideoxycholate, lithocholate, alpha-muricholate, beta-muricholate, omega-muricholate, murideoxycholate, beta-phocaecholate, taurochenodeoxycholate, taurocholate, taurodeoxycholate, ursocholate, ursodeoxycholate, and vulpecholate and a combination thereof. Bile acid is a conjugate acid of bile acid anion. A “conjugate base of a bile acid” may be obtained following dissolution of a bile acid or bile salt in an aqueous solution. For example, taurodeoxycholate (TDC) may be obtained following dissolution of taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7) or sodium taurodeoxycholate (CAS No.: 1180-95-6) in an aqueous solution. A bile salt may be a salt of a bile acid, such as for example, a sodium salt of a bile acid.

“Adsorption of bile acids to Sucralfate” used in the context of the invention refers to a binding interaction between Sucralfate as a solid and bile acid or bile salt dissolved in a solution.

“Binding of Sucralfate to bile acid” may be binding of Sucralfate to bile acid or to a conjugated bile acid.

The term “effective amount” means an amount of a compound/composition according to the present invention effective in producing the desired therapeutic effect.

The term “about” when used in connection with percentages or values means+/−10% or less, preferably +/−1%. As used, “about” may refer to errors associated with measurements and variation observed, e.g., between assays.

The “upper GI tract” refers to the portion closest to the stomach and includes the esophagus, stomach and duodenum.

The term “treating” a disease or condition, means to manage a disease or condition with the pharmaceutical formulation of the invention. Treatment can decrease the symptoms of a disease or condition, reduce the severity of a disease or condition, alter the course of disease progression or condition, ameliorate and/or cure a disease or condition. The disease or condition may include but not limited to an ulcer, such as active duodenal ulcer and peptic ulcer disease (PUD), gastritis due to gastroesophageal disease (GERD), irritable bowel syndrome (IBS) non-erosive reflex disorder (NERD) and functional dyspepsia (FD).

The term “bioequivalence” refers to the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study. It would be clear to one skilled in the art that in some embodiments, the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives may become available at the site of drug action when administered at the same amount or dose under similar conditions.

The term “in vitro bioequivalence” refers to an in vitro approach to demonstrating bioequivalence through a combination of in vitro binding studies and bioassays that support the postulated modes of action. For example, a test product formulation may be developed which is qualitatively and quantitatively the same or essentially the same as the RLD with respect to active and inactive ingredients. The critical material attributes and process parameters are optimized until similarity of the test product in formulation function and in the bioassays with the RLD product is achieved. With greater sampling (within a particular lot and between multiple lots of a test product and/or RLD product), there is greater confidence that the “in vitro bioequivalence” is reflective of the in vivo “bioequivalence” to ensure and establish a product efficacy and a therapeutic equivalence without resorting to a three-arm, placebo-controlled, in-vivo clinical endpoint study.

METHODS OF THE INVENTION

The invention provides a method of determining in vitro bioequivalence of a Sucralfate suspension sample to a Sucralfate Suspension RLD, based on a combination of in vitro binding studies and bioassays that support the postulated modes of action. The method comprises in vitro binding of Sucralfate to bile acid(s) and in vitro bioassay for Sucralfate-mediated inhibition of cell damage by a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 in a cell culture system. Comparison of the effectiveness of a Sucralfate Suspension sample to that of a Sucralfate Suspension RLD in the combination of in vitro binding study and bioassay is used to establish in vitro bioequivalence of the Sucralfate Suspension sample to the Sucralfate Suspension RLD.

The present invention provides, for the first time, methods for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprising contacting the sucralfate suspension sample with a bile acid, bile salt and/or conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD.

The present invention additionally comprises contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system, quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD.

The reference values for the sucralfate suspension RLD to be used in comparing the sucralfate-bile interaction are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; wherein sucralfate-bile interaction is interaction between sucralfate and bile acid, bile salt and/or conjugated bile acid or a combination thereof; wherein the reference values of the sucralfate suspension RLD to be used in comparing the cell damage are derived from a set of values obtained from quantifying the reduction in cell damage for two (2) or more lots of the sucralfate suspension RLD; and wherein the sucralfate suspension sample is to have said in vitro bioequivalence to a sucralfate suspension RLD when the values determined for the sucralfate suspension sample are within the reference values for the sucralfate suspension RLD.

In one embodiment of the invention, the method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprises: step a) contacting the sucralfate suspension sample with a bile acid, bile salt and/or a conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD; and step b) contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system, quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD, wherein the cell culture system of step (b) comprises mammalian epithelial cells; and wherein cell damage comprises loss of the cell monolayer's transepithelial electrical resistance (TEER). Further, in this embodiment, the reference values for the sucralfate suspension RLD in step (a) are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; wherein sucralfate-bile interaction is interaction between sucralfate and bile acid, bile salt and/or conjugated bile acid or a combination thereof; wherein the sucralfate suspension sample is to have said in vitro bioequivalence to a sucralfate suspension RLD when the values determined in steps (a) and (b) for the sucralfate suspension sample are within the reference values in steps (a) and (b) for the sucralfate suspension RLD, respectively.

In yet another specific embodiment of the invention, the method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprises: step a) contacting the sucralfate suspension sample with a bile acid, bile salt and/or a conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD; and b) contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD, wherein the cell culture system of step (b) comprises mammalian epithelial cells; and wherein cell damage comprises loss of the cell monolayer's transepithelial electrical resistance (TEER). Additionally, in this embodiment, the reference values for the sucralfate suspension RLD in step (a) are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; wherein sucralfate-bile interaction is interaction between sucralfate and bile acid, bile salt and/or conjugated bile acid or a combination thereof; and wherein the sucralfate suspension sample is to have said in vitro bioequivalence to a sucralfate suspension RLD when the values determined in steps (a) and (b) for the sucralfate suspension sample are within the reference values in steps (a) and (b) for the sucralfate suspension RLD, respectively, wherein the reference values in steps (a) and (b) for the sucralfate suspension RLD are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence includes no less than about 80% of the values in steps (a) and (b) for the sucralfate suspension sample to be within the range of reference values in steps (a) and (b), respectively.

Additionally, in a further embodiment, the method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprises: step a) contacting the sucralfate suspension sample with a bile acid, bile salt and/or a conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD; and step b) contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD, wherein the cell culture system of step (b) comprises mammalian epithelial cells; and wherein cell damage comprises α-loss of the cell monolayer's transepithelial electrical resistance (TEER). In this embodiment, the reference values for the sucralfate suspension RLD in step (a) are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; wherein sucralfate-bile interaction is interaction between sucralfate and bile acid, bile salt and/or conjugated bile acid or a combination thereof; and wherein the sucralfate suspension sample is to have said in vitro bioequivalence to a sucralfate suspension RLD when the values determined in (a) and (b) for the sucralfate suspension sample are within the reference values in steps (a) and (b) for the sucralfate suspension RLD, respectively, wherein the reference values in steps (a) and (b) for the sucralfate suspension RLD are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence requires the values in steps (a) and (b) for the sucralfate suspension sample to be within the range of reference values in steps (a) and (b), respectively.

Further, in another embodiment, the method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprises: step a) contacting the sucralfate suspension sample with a bile acid, bile salt and/or a conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD; and step b) contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD; wherein the cell culture system of step (b) comprises mammalian epithelial cells; and wherein cell damage comprises a loss of the cell monolayer's transepithelial electrical resistance (TEER). In this embodiment, the reference values for the sucralfate suspension RLD in step (a) are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; wherein sucralfate-bile interaction is interaction between sucralfate and bile acid, bile salt and/or conjugated bile acid or a combination thereof; and wherein the sucralfate suspension sample is to have said in vitro bioequivalence to a sucralfate suspension RLD when the values determined in steps (a) and (b) for the sucralfate suspension sample are within the reference values in steps (a) and (b) for the sucralfate suspension RLD, respectively, and wherein the sucralfate suspension sample is said to have in vitro bioequivalence to a sucralfate suspension RLD when (i) the value determined in quantifying the sucralfate-bile interaction of the sucralfate suspension sample with a bile acid, bile salt and/or conjugated bile acid or a combination thereof is within the 90% confidence interval range of a set of SCFeq values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; (ii) TEER values determined in quantifying the cell damage of the sucralfate suspension sample with a cell culture system is within the 90% confidence interval range of the set of values obtained from quantifying the cell damage for 2 or more lots of the sucralfate suspension RLD; (iii) ratio of mean of the values (Mean of the Test/Mean of the RLD) determined in quantifying the sucralfate-bile interaction of the sucralfate suspension sample with a bile acid, bile salt and/or bile conjugate base or a combination thereof to the mean of the set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension reference listed drug is within the range of about 0.80 to 1.25; and (iv) ratio of mean of the values determined in quantifying the cell damage of the sucralfate suspension sample with a cell culture system to the mean of the set of values obtained from quantifying the cell damage for 2 or more lots of the sucralfate suspension reference listed drug is within the range of about 0.80 to 1.25; and (v) the 90% confidence interval is a range of values within about 1.5 standard deviations about the mean of values measured for two or more lots of the Sucralfate suspension RLD.

Suitable examples of bile acids include, but are not limited to, allocholic acid (3alpha,7alpha,12alpha-trihydroxy-5alpha-cholanoic acid; CAS: 2464-18-8), 5alpha-deoxycholic acid (3alpha,12alpha-dihydroxy-5alpha-cholan-24-oic acid), bitocholic acid, chenodeoxycholic acid (3alpha,7alpha-dihydroxy-5beta-cholan-24-oic acid; CAS: 474-25-9), cholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 81-25-4), deoxycholic acid (3alpha,12alpha-dihydroxy-5beta-cholanic acid; CAS: 83-44-3), glycochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid; CAS No.: 640-79-9), glycocholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oylglycine; CAS: 475-31-0), hyocholic acid (3alpha,6alpha,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 547-75-1), hyodeoxycholic acid (3α,6α-Dihydroxy-5β-cholan-24-oic acid; CAS: 83-49-8), isochenodeoxycholic acid (3beta,7alpha-dihydroxy-5beta-cholanic acid; CAS: 566-24-5), 3beta,12alpha-Dihydroxy-5beta-cholanoic acid (CAS: 570-63-8), isolithocholic acid (3beta-Hydroxy-5beta-cholan-24-oic acid; CAS: 1534-35-6), isoursodeoxycholic acid (3beta,7beta-dihydroxy-5beta-cholan-24-oic acid; CAS: 78919-26-3), 12-epideoxycholic acid, lithocholic acid (3alpha-hydroxy-5beta-cholanic acid; CAS: 434-13-9), alpha-muricholic acid (3alpha,6beta,7alpha-trihydroxy-5beta-cholan-24-oicacid; CAS: 2393-58-0), beta-muricholic acid (3alpha,6beta,7beta-trihydroxy-5beta-cholan-24-oic acid; CAS: 2393-59-1), omega-muricholic acid, murideoxycholic acid (3alpha,6beta-dihydroxy-5beta-cholanic acid), beta-phocaecholic acid (CAS: 105369-89-9), taurochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CAS No.: 516-35-8), taurocholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CAS No.: 81-24-3), taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7), ursocholic acid (3alpha,7beta,12alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 2955-27-3), ursodeoxycholic acid (3alpha,7beta-dihydroxy-5beta-cholan-24-oic acid; CAS: 128-13-2), and vulpecholic acid (1alpha,3alpha,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 107368-95-6) or a combination thereof.

In one embodiment, the bile salt is salt of a bile acid. In another embodiment, the bile salt is sodium salt of a bile acid. In yet another embodiment, the bile salt is potassium salt of a bile acid.

In one embodiment, the bile acid may be any one or more of a glycocholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oylglycine; CAS No.: 475-31-0), glycochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid; CAS No.: 640-79-9), and taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7) or a combination thereof. In another embodiment, the bile acid is or comprises a taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7).

In one embodiment, the bile acid is a conjugate base of a bile acid. In another embodiment, the conjugate base of a bile acid may be any of glycocholate, glycochenodeoxycholate, taurochenodeoxycholate and taurodeoxycholate or a combination thereof. In yet another embodiment, the conjugate base of a bile acid is or comprises taurodeoxycholate.

In one embodiment, the bile acid is an unconjugated bile acid or bile acid not conjugated to glycine or taurine. In another embodiment, the unconjugated bile acid may be any one or more of allocholic acid, allodeoxycholic acid, bitocholic acid, chenodeoxycholic acid, cholic acid, deoxycholic acid, hyodeoxycholic acid, isochenodoxycholic acid, 3β,12α-dihydroxy-5β-cholanoic acid, isolithocholic acid, isoursodeoxycholic acid, 12-epideoxycholic acid, lithocholic acid, α-muricholic acid, β-muricholic acid, ω-muricholic acid, murideoxycholic acid, β-phocaecholic acid, ursocholic acid, ursodeoxycholic acid, vulpecholic acid, and hyocholic acid.

In one embodiment, the bile acid is a conjugated bile acid. In another embodiment, the conjugated bile acid may be any one or more of glycocholate, glycochenodeoxycholate, taurochenodeoxycholate and taurodeoxycholate or a combination thereof. In yet another embodiment, the conjugated bile acid is or comprises taurodeoxycholate.

In one embodiment, the conjugated bile acid is any one or more of allocholic acid, allodeoxycholic acid, bitocholic acid, chenodeoxycholic acid, cholic acid, deoxycholic acid, hyodeoxycholic acid, isochenodoxycholic acid, 3β,12α-dihydroxy-5β-cholanoic acid, isolithocholic acid, isoursodeoxycholic acid, 12-epideoxycholic acid, lithocholic acid, α-muricholic acid, β-muricholic acid, ω-muricholic acid, murideoxycholic acid, β-phocaecholic acid, ursocholic acid, ursodeoxycholic acid, vulpecholic acid or hyocholic acid, conjugated to a glycine or a taurine.

In one embodiment, the bile salt is a bile acid salt or salt of a conjugate base of a bile acid. In another embodiment, the bile salt comprises a bile acid salt or comprises a conjugate base of a bile acid and a cation includes, but is not limited to, an alkali metal, alkaline earth metal, transition metal, other metal, ammonium, amine and quaternary ammonium. Suitable examples of the alkali metal include, but are not limited to, lithium, sodium, potassium, rubidium and cesium. Suitable examples of the alkaline earth metal include, but are not limited to, beryllium, magnesium, calcium, strontium and barium. Suitable examples of the transition metal include, but are not limited to, chromium, molybdenum, manganese, iron, cobalt, nickel, copper, silver, gold, zinc and cadmium. Suitable examples of the amine include, but are not limited to, methylamine, methoxylamine, methylenediamine, bromoethylamine, chloroethylamine, fluoroethylamine, dimethylamine, ethylenediamine, diethylamine, cystamine, aniline, nitro-benzenediamine, phenylenediamine, tris(2-chloroethyl)amine, tromethamine, ethanolamine, diethanolamine and triethanolamine. Suitable examples of the quaternary ammonium include, but are not limited to, tetramethylammonium, tetrabutylammonium, tetraethylammonium, triethylmethylammonium and tributylmethylammonium. In one embodiment, the bile salt or bile acid salt is a sodium salt of a bile acid. In another embodiment, the bile salt or bile acid salt is a potassium salt of a bile acid. In another embodiment, the bile salt or bile acid salt is or comprises a sodium taurodeoxycholate (e.g., CAS No.: 1180-95-6).

In another embodiment, the bile salt comprises a conjugated bile acid and a cation includes, but is not limited to, an alkali metal, alkaline earth metal, transition metal, other metal, ammonium, amine and quaternary ammonium. Suitable examples of the alkali metal include, but are not limited to, lithium, sodium, potassium, rubidium and cesium. Suitable examples of the alkaline earth metal include, but are not limited to, beryllium, magnesium, calcium, strontium and barium. Suitable examples of the transition metal include, but are not limited to, chromium, molybdenum, manganese, iron, cobalt, nickel, copper, silver, gold, zinc and cadmium. Suitable examples of the amine include, but are not limited to, methylamine, methoxylamine, methylenediamine, bromoethylamine, chloroethylamine, fluoroethylamine, dimethylamine, ethylenediamine, diethylamine, cystamine, aniline, nitro-benzenediamine, phenylenediamine, tris(2-chloroethyl)amine, tromethamine, ethanolamine, diethanolamine and triethanolamine. Suitable examples of the quaternary ammonium include, but are not limited to, tetramethylammonium, tetrabutylammonium, tetraethylammonium, triethylmethylammonium and tributylmethylammonium.

In one embodiment, the sucralfate-bile interaction is binding of bile acid, bile salt or conjugated bile acid or a combination thereof by sucralfate. Merely by way of example, the binding may be in vitro equilibrium binding. In another example, the binding may be in vitro kinetic binding. In yet another example, the binding may be adsorption of bile acid, bile salt or conjugated bile acid or a combination thereof to sucralfate. Merely by way of example, the interaction or association may delay the migration of bile acid, bile salt or conjugated bile acid or a combination thereof in the presence of sucralfate.

In one embodiment, the cell culture system comprises a tissue culture cell. In a further embodiment, the tissue culture cell comprises a mammalian cell. In an embodiment, the mammalian cell includes, but is not limited to, human cell, monkey cell, ape cell, mouse cell, rat cell, hamster cell, rabbit cell, guinea pig cell, cow cell, swine cell, dog cell, horse cell, cat cell, goat cell, and sheep cell. In a preferred embodiment, the mammalian cell is a human cell or a dog cell.

In another embodiment, the mammalian cell is a mammalian epithelial cell. In a further embodiment, the mammalian cell is derived from the gastrointestinal tract including, but not limited to, the oesophagus, stomach, duodenum, small intestine, caecum, colon, rectum, liver, gall baldder, spleen, biliary tree, pancreas, and kidney. In a preferred embodiment, the mammalian cell is a kidney cell (e.g., a kidney cell such as Madin-Darby Canine Kidney (MDCK) cells). In a more preferred embodiment, the mammalian cell is an epithelial cell (e.g., a human epithelial cell). In another embodiment, the mammalian epithelial cell is a mammalian intestinal epithelial cell and/or gastrointestinal cell. In a most preferred embodiment, the human epithelial cell is a human intestinal epithelial cell and/or gastrointestinal cell. In still a further embodiment, the human intestinal epithelial cell is any of Caco-2, T-84 intestinal epithelial cells, Madin Darby canine kidney (MDCK) cells and small-intestine cell lines from fetal and neonatal rats (IEC and REI). In still a further embodiment, the human intestinal epithelial cell is Caco-2.

Suitable examples of NSAIDs include, but are not limited to, aceclofenac, acetyl salicylic acid, choline magnesium salicylate, clonixin, diflunisal, magnesium salicylate, salicyclic acid, salicylate, salsalate, sodium salicylate, dexibuprofen, dexketoprofen, diclofenac, droxicam, etodolac, fenoprofen, flufenamic acid, flurbiprofen, indomethacin, isoxicam, ketoprofen, ketorolac, lomoxican, loxoprofen, meclofenamate, meclofenamic acid, mefenamic acid, meloxicam, naproxen, nabumetone, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tolfenamic acid, tolmetin, ibuprofen, Cox-2 inhibitors and tramadol. In one embodiment, the NSAID is indomethacin.

In one embodiment, the nonselective inhibitor of cyclooxygenase (COX) 1 and 2 include, but are not limited to, one or more of aspirin, diclofenac, ibuprofen, naproxen, mefenamic acid, indomethacin, ketoprofen and piroxicam and equivalents thereof.

Suitable examples of cell damage include, but are not limited to, cell death, necrosis, apoptosis, gastric lesion, loss in mucosal barrier, loss in barrier property of intestinal epithelium, decrease in transmucosal electrical potential difference, and loss of cell monolayer's transepithelial electrical resistance (TEER). In one embodiment, cell damage comprises loss of cell monolayer's transepithelial electrical resistance (TEER). In another embodiment, cell damage is loss of cell monolayer's transepithelial electrical resistance (TEER).

In one embodiment, the values for quantifying the sucralfate-bile interaction and quantifying the cell damage for the sucralfate suspension sample are obtained from multiple measurements of a single lot of sucralfate suspension sample or measurements from multiple lots of sucralfate suspension sample or a combination thereof. In another embodiment, the values for the sucralfate suspension RLD to be used in comparing the sucralfate-bile interaction and comparing the cell damage are obtained from two or more lots of sucralfate suspension RLD. In yet another embodiment, the reference values for the sucralfate suspension RLD to be used in comparing the sucralfate-bile interaction and comparing the cell damage are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence includes no less than about 80% of the values for the sucralfate suspension sample to be within the range of reference values when comparing the sucralfate-bile interaction and the cell damage, respectively.

In yet another embodiment, the reference values for the sucralfate suspension RLD to be used in comparing the sucralfate-bile interaction and comparing the cell damage are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence includes no less than about 90% of the values for the sucralfate suspension sample to be within the range of reference values when comparing the sucralfate-bile interaction and the cell damage, respectively. In one embodiment, the reference values for the sucralfate suspension RLD in comparing sucralfate-bile interaction and comparing cell damage are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence requires substantially or nearly all of the values for the sucralfate suspension sample to be within the range of reference values in sucralfate-bile interaction and cell damage, respectively. In one example, the reference values for comparing the sucralfate-bile interaction for the sucralfate suspension RLD is the range of about 7 to about 17 mg/mL including e.g., 7.08 to about 16.14 mg/mL, wherein the reference values for the sucralfate suspension RLD are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD. In another example, the reference values for comparing the cell damage for the sucralfate suspension RLD is a TEER range of about 23 to about 75% including e.g., about 23.3% to about 74.8%, wherein the reference values of the sucralfate suspension RLD are derived from a set of values obtained from quantifying the cell damage for 2 or more lots of the sucralfate suspension RLD. In yet another example, the reference values for comparing the cell damage for the sucralfate suspension RLD has a TEER range of about 64% to about 93% including e.g., about 64.3% to about 92.5%. Measurements are conducted under well-controlled conditions so as to permit comparison of the measured results for sucralfate suspension RLD and sucralfate suspension sample.

In one embodiment, the values are statistical means. In a further embodiment, the means of the values determined for the sucralfate suspension sample fall within two standard deviations or three standard deviations of the means determined for the set of reference values set for the sucralfate RLD. In another embodiment, the means of the values determined for the sucralfate suspension sample are within one standard deviation of the means determined for the set of reference values set for the sucralfate RLD.

In one embodiment, the sucralfate suspension sample is said to have in vitro bioequivalence to a sucralfate suspension RLD when (i) the value determined in quantifying the sucralfate-bile interaction of the sucralfate suspension sample with a bile acid, bile salt and/or conjugated bile acid or a combination thereof is within the range of the set of SCFeq values obtained from quantifying the sucralfate-bile interaction for 2 or more lots (including 3 or more; 4 or more; or 5 or more lots) of the sucralfate suspension RLD; (ii) the TEER values determined in quantifying the cell damage of the sucralfate suspension sample with a cell culture system is within the set of values obtained from quantifying the cell damage for 2 or more lots of the sucralfate suspension RLD; (iii) ratio of mean of the values (Mean of the Test/Mean of the RLD) determined in quantifying the sucralfate-bile interaction of the sucralfate suspension sample with a bile acid, bile salt and/or bile conjugate base or a combination thereof to the mean of the set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots (including 3 more; 4 or more; or 5 or more lots) of the sucralfate suspension reference listed drug; and (iv) ratio of mean of the values determined in quantifying the cell damage of the sucralfate suspension sample with a cell culture system to the mean of the set of values obtained from quantifying the cell damage for 2 or more lots of the sucralfate suspension reference listed drug is within the range of about 0.80 to 1.25; and (v) the about 90% confidence interval on the difference in product means falls entirely within the range from −EAC to +EAC; where EAC is the Equivalence Acceptance Criterion selected as EAC=1.5×S where S is the square root of the total variance for RLD The invention further provides a method for obtaining a sucralfate suspension sample that is a bioequivalent of a sucralfate suspension RLD by determining in vitro bioequivalence.

The invention further provides a method for reducing or alleviating the symptoms associated with an ulcer in a patient suffering from the ulcer comprising administering to the patient an effective amount of any one or more of the sucralfate suspension samples thereby reducing or alleviating the symptoms associated with an ulcer in the patient.

In accordance with the practice of the invention, the delay in migration of bile acid, bile salt or conjugated bile acid or a combination thereof in the presence of sucralfate may comprise a decrease in diffusion of bile acid, bile salt or conjugated bile acid or a combination thereof in the presence of sucralfate. For example, the bile salt may be or comprise taurodeoxycholate (TDC).

In another embodiment of the invention, the delay in migration may comprise a delay in migration or diffusion of taurodeoxycholate (TDC) in the presence of sucralfate. In a further embodiment, the delay in migration may comprise a delay observed additionally in the presence of a protein or a combination of proteins. Examples of suitable proteins include, but are not limited to albumin, fibrinogen, hemoglobin, casein, and enzymes, such as trypsin and gastrin, or a combination thereof. Merely by way of example, the protein or a combination of proteins is isolated from a subject or recombinantly produced.

Albumin may be serum albumin or recombinant albumin. Suitable examples of serum albumin include, but are not limited to, bovine serum albumin, human serum albumin, ovine serum albumin, porcine serum albumin, caprine serum albumin, leporine serum albumin, equine serum albumin, canine serum albumin, feline serum albumin, rodent serum albumin, and monkey serum albumin or a combination thereof. Suitable examples of recombinant albumin include, but are not limited to, bovine albumin, human albumin, ovine albumin, porcine albumin, caprine albumin, leporine albumin, equine albumin, canine albumin, feline albumin, rodent albumin, and monkey serum or a combination thereof.

In another embodiment of the invention, the combination of proteins are or may comprise proteins in plasma exudate at a wound site, proteins in mammalian serum, proteins in human serum or a combination thereof.

Additionally, in one embodiment, the delay in migration or diffusion of taurodeoxycholate (TDC) in the presence of sucralfate additionally comprises human serum albumin. Merely by way of example, the delay in migration or the decrease in diffusion may comprise determining a permeability value between two compartments. For example, the permeability value may be an apparent permeability value for bile acid, bile salt or conjugated bile acid or a combination thereof determined from concentration of bile acid, bile salt or conjugated bile acid or a combination for two adjacent compartments.

Also, in an embodiment of the invention, the delay in migration of bile acid, bile salt or conjugated bile acid or a combination thereof, additionally may comprise formation of a film or barrier comprising sucralfate. For example, the film or barrier may additionally comprise a protein or a combination of proteins. In another embodiment, the film or barrier may additionally comprise serum albumin or recombinant albumin. For example, the serum albumin or recombinant albumin may be human serum albumin or recombinant human albumin.

Also in accordance with the practice of the invention, in another embodiment, the film or barrier may be between one compartment and a second compartment or separates one compartment from a second compartment. For example, the compartment may comprise a liquid compartment.

The invention also provides in the method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD described above, further comprising, in addition to any of a step a) and step b) above, an additional step, namely, step (c) which is the step of contacting the sucralfate suspension sample with a solution comprising a bile acid, bile salt and/or a conjugated bile acid or a combination thereof, and albumin, so as to permit formation of a sucralfate film or barrier between two compartments, quantifying a delay in migration or diffusion of the bile acid, bile salt and/or a conjugated bile acid or a combination thereof from one compartment to the second compartment and comparing said values to reference values for the sucralfate suspension RLD. In this embodiment, the reference values for the sucralfate suspension RLD in step (c) may be derived from a set of values obtained from quantifying the delay in migration or diffusion of bile acid, bile salt and/or a conjugated bile acid or a combination thereof, for 2 or more lots of the sucralfate suspension RLD. Further, in an embodiment of this invention, the delay in migration or diffusion comprises a sucralfate film or barrier between two compartments. Additionally, in one embodiment, the sucralfate suspension sample said to have in vitro bioequivalence to a sucralfate suspension RLD is further claimed or verified to have said in vitro bioequivalence to a sucralfate suspension RLD when the values further determined in step (c) for the sucralfate suspension sample are within the reference values in step (c) for the sucralfate suspension RLD.

Also, in an embodiment of the invention, quantifying a delay in migration or diffusion of the bile acid, bile salt and/or a conjugated bile acid or a combination thereof from one compartment to the second compartment may comprise a permeability value. For example, the permeability value may be an apparent permeability value or Papp. In another embodiment, the reference values in step (c) for the sucralfate suspension RLD may be experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence includes no less than about 80% of the values in step (c) for the sucralfate suspension sample to be within the range of reference values in step (c).

Merely by way of example, the reference values in step (c) include no less than about 90% of the values in step (c) for the sucralfate suspension sample to be within the range of reference values in step (c). In another embodiment, in the reference values in step (c) for the sucralfate suspension RLD are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence requires substantially or nearly all of the values in step (c) for the sucralfate suspension sample to be within the range of reference values in step (c). In one embodiment, the reference values of step (c) for the sucralfate suspension RLD may be in the range of about 10.76×10⁶ cm/s to about 14.12×10⁶ cm/s for Papp values.

In accordance with the practice of the invention, the adsorption of bile acid, bile salt or conjugated bile acid or a combination thereof to sucralfate may be described by Langmuir equation. For example, a Langmuir equation may be:

x/m=k ₁ *k ₂ *C _(eq)/(1+k ₁ *C _(eq))

or upon rearranging is:

C _(eq)/(x/m)=1/(k ₁ *k ₂)+C _(eq) /k ₂

Where:

-   -   C_(eq) (mM)=concentration of TDC remaining in the supernatant         after incubation     -   x/m (mmol/g)=amount of TDC (mmol) bound per the amount of         sucralfate (g)     -   k₁ (1/mM or L/mmol)=affinity constant     -   k₂ (mmol/g)=Langmuir capacity constant.

In one embodiment of the invention, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD in (a) comprises determining affinity constant, k₁, and/or Langmuir capacity constant, k₂, e.g., using a Langmuir equation. In a specific embodiment, the interaction is or comprises adsorption of TDC to sucralfate. In yet another specific embodiment, the reference values of step (a) for the adsorption of TDC to sucralfate is or comprises Langmuir capacity constant, k₂, in the range of about 135 μmol (TDC)/g (sucralfate) to about 201 μmol (TDC)/g (sucralfate). In a further embodiment, the range of the Langmuir capacity constant, k₂, may change as additional lots of RLDs are assayed and/or different condition are used for the binding assay. In another embodiment, the Langmuir capacity constant, k₂, may be determined for other bile acid, bile salt and/or conjugated bile acid or a combination thereof. In yet another embodiment, the reference values of step (a) for the adsorption of TDC to sucralfate is or comprises an affinity constant, k₁, of the Langmuir equation or k₂, the Langmuir capacity constant, and determining in vitro bioequivalence of a sucralfate suspension sample comprises or additionally comprises comparing said values for the sucralfate suspension sample to reference values for the sucralfate suspension RLD. In a further embodiment of the invention, the reference values in a step (a) (of the invention) for the sucralfate suspension RLD are experimentally determined values characterized by a minimum and a maximum value and in vitro bioequivalence which includes no less than about 80% of the values in step (a) for the sucralfate suspension sample to be within the range of reference values in (a). In yet an additional embodiment, the reference values in step (a) for the sucralfate suspension RLD includes no less than about 90% of the values in step (a) for the sucralfate suspension sample to be within the range of reference values in step (a). Further still, in another embodiment of the invention, the reference values in step (a) requires substantially or nearly all of the values in (a) for the sucralfate suspension sample to be within the range of reference values in step (a).

Advantages of the Invention

Bioequivalence (BE) assessment is essential for generic drug development and availability of any interchangeable product. The requirements for a BE study vary greatly from product to product for various reasons. The underlying scientific basis for the BE assessment for the generic drug product is that it should be safe and as effective as the reference drug product.

For the majority of drug products, the requirements of a BE study are both ethical and scientifically appropriate in accurately deducing a product's therapeutic equivalence. In the case of Sucralfate oral suspension, as of today there is no guidance from the FDA for assessing BE for Sucralfate.

For Sucralfate, a site of action is local in the GI tract. The drug product dissolution and transit controls the presentation of drug to the site of action and the drug's plasma concentration is downstream from the site of action and irrelevant to the clinical effect. It also has very low systemic availability and is not detected in plasma. As such, plasma levels of Sucralfate cannot be used to establish bioequivalence of a Sucralfate Suspension sample to a Sucralfate Suspension RLD.

The current invention provides an advantage by providing a method for establishing therapeutic equivalence of a pharmaceutically equivalent Sucralfate Suspension sample against a Sucralfate Suspension RLD. It provides a more accurate, sensitive and reproducible method than previously proposed and subsequently withdrawn method based on clinical endpoint. Not only is the in vitro bioequivalence method of the invention based on biochemistry and bioassay more accurate, sensitive and reproducible but it is also ethical, expeditious, robust and cost effective than a method based on clinical endpoint.

The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the invention.

EXAMPLES Example 1

Bile-acid binding assessment using taurodeoxycholate (TDC): Optimized assay conditions are as follows: The TDC concentration was 2.5 mM. The Sucralfate concentrations were at 0.5, 1, 2, 4, 8, 16, 32 and 50 mg/mL. TDC is incubated with different concentrations of Sucralfate at pH 1.5 and 37° C. for 60 minutes; following incubation, each sample is spun at 12,500×g for 30 min at room temperature to remove TDC bound to Sucralfate. Concentration of free TDC is determined by collecting the supernatant and analyzing the presence of TDC by LC-MS/MS detection analytical method.

Sucralfate adsorption of bile acids and salts-agents in erosive mucosal injury and ulceration. The gastric mucosal barrier is considered to play an important role in protecting the gastric mucosa from the destructive effects of several luminal agents including H⁺ ions diffusing from the lumen into the mucosa (back-diffusion). This barrier consists of mainly three components: an epithelial cell lining where the cells are bound by tight junctions that repel harsh fluids that may harm the lining, a mucus covering which protects the mucosa from auto-ingestion by pepsin and from erosion by acids and other caustic materials ingested, and bicarbonate ions which act to neutralize harsh acids [16]. The breakdown of this barrier has been associated with erosive mucosal injury and ulceration induced by factors such as stress and endogenous compounds including bile acids, which is one of the major components of bile (67%) along with phospholipids (22%), proteins (4.5%), cholesterol (4%) and bilirubin (0.3%) [17]. Bile salts are composed of the salts of four different kinds of free bile acids (cholic, deoxycholic, chenodeoxycholic and lithocholic acids) and each of these acids combines with glycine or taurine to form conjugated bile acids and salts. Bile acids are considered to be one of the important factors in the pathogenesis of ulcer disease, and the binding of bile acids to antacids has been considered to play an important role in the therapeutic action in the treatment of ulcer [11]. The exact mechanism by which the bile salts disrupt the gastric mucosal barrier is unknown, and a couple of potential mechanisms postulated are the mucosal uptake of bile salts and dissolution of mucosal lipids by the detergent action of intraluminal bile salt micelles [18]. In the presence of acid, Sucralfate releases aluminum, acquires a strong negative charge and binds electrostatically to any positively charged chemical groups in its environment, including proteins, peptides, and drugs. Adsorption of bile acids to Sucralfate along with several aluminum-containing antacids has been examined thoroughly and widely published (19, 27). Potent bile acid binding properties of Sucralfate, which are pH-dependent, have been reported [11]. Therefore, for all of the reasons stated above, it was deemed critical to compare the bile acid binding capacity of the RLD product with that of the test product to be able to compare the protective action of Sucralfate using both products.

Experiments were then executed as per the assay eligibility determination process below. The results of each step are discussed below.

Step 1: Assay Development

-   -   1. Determination of analytical sensitivity for determination of         TDC concentrations     -   2. Bile acid selection     -   3. Optimum pH condition selection for bile acid binding     -   4. Evaluation of TDC binding to Sucralfate at varied TDC         concentrations for selection of optimal concentration of TDC for         comparative studies     -   5. Incubation duration     -   6. Sample preparation procedures

Step 2: Assay Qualification

-   -   1. Evaluate assay reproducibility     -   2. Determine assay selectivity by evaluating the dose-response         of Sucralfate binding to TDC     -   3. Evaluate the range of Sucralfate binding to bile acids using         multiple lots of RLD.

Step 3: Assay Performance

-   -   1. Evaluate binding of TDC for the initial prototype test         formulation (Test_50) and compare with that of the reference         formulation(s)     -   2. Evaluate binding of TDC for the further optimized test         formulation (Test_66) and compare with that of the reference         formulation(s)

Results and Discussion: Step 1: Assay Development:

1. Determination of Analytical Sensitivity for Determination of TDC Concentrations:

Multiple standard curves were generated to determine and confirm the analytical sensitivity for the detection of TDC using LC-MS/MS methodology for detection. The range of TDC concentrations evaluated was 0-40 μM. The results, as illustrated in FIG. 1, showed that the standard curves generated under these conditions were consistently reproducible and the assay was sufficiently sensitive to detect TDC.

2. Bile Acid Selection:

Several conjugated bile acids such as glycocholate (GC), glycochenodeoxycholate (GCDC), glycodeoxycholate (GDC), taurocholate (TC), taurodeoxycholate (TDC), and taurochenodeoxycholate (TCDC) among others, have been reported to be present in human gastric and duodenal aspirates [11]. Graham et al. (1984) reported that Sucralfate had the maximum binding capacity to TDC and TCDC (with binding capacity to TDC being greater as compared to TCDC). Therefore, both TDC and TCDC were selected for the in-vitro evaluation of bile acid binding properties of Sucralfate. TDC was then chosen as the bile acid for further assessments based on the findings that thebinding capacity of Sucralfate was greater for TDC as compared to TCDC.

3. Optimum pH Condition Selection for Bile Acid Binding and Evaluation of TDC Binding to Sucralfate at Varied TDC Concentrations for Selection of Optimal Concentration of TDC for Comparative Studies:

Several aspects of the assay design such as assay pH, duration, TDC concentration, and sample preparation procedures were adapted from various references such as the Graham et al. and Lipsett et al. publications and findings were confirmed and/or further optimized [11,19]. Both pH- and concentration-dependent binding of Sucralfate to various bile acids has been reported, with much greater bile acid binding by Sucralfate at lower pH [11]. Additionally, concentrations used for various bile acid(s) in these studies varied from 1 to 10 mM, and concentrations for TDC specifically ranged from 1 to 5 mM.

Based on the abundant information available for assay conditions in the literature, 1) TDC binding to Sucralfate was evaluated at pH 1.5 and 4.5 and 2) TDC binding to Sucralfate was evaluated at 3 different TDC concentrations: 1, 2.5 and 5 mM for using Sucralfate concentration range of 2 to 50 mg/mL using an assay duration of 60 minutes.

Based on the results of the optimization experiments, the assay conditions were then fixed as outlined above. Each assay condition was chosen because it resulted in maximum binding of Sucralfate at that particular condition allowing it to be the most discriminatory condition for the purpose of comparing the RLD and test products. A concentration of 2.5 mM TDC was selected as it appeared to have the greatest dynamic range of % bound for the range of Sucralfate concentrations evaluated FIG. 2. An assay pH of 1.5 was selected based on several references that reported maximum Sucralfate binding to bile acids at lower pH which was also confirmed by results generated in house.

Step 2: Assay Qualification:

1. Evaluation of Assay Reproducibility:

Once the assay conditions were optimized, reproducible assay performance was confirmed by evaluating the Sucralfate binding to TDC in replicates using the optimized conditions using one lot of RLD product. The results confirmed the reproducibility of the assay using the fixed experimental conditions as tabulated in Table 1.

TABLE 1 Evaluation of assay reproducibility and dose-response of Sucralfate binding to TDC using one representative lot of RLD product (RLD_04) Sucralfate Concentration RLD_04 TDC (mg/ml) binding SD 50 75% 12%  32 74% 9% 16 62% 6% 8 20% 17%  4  5% 7% 2  2% 3%

2. Determination of Assay Selectivity by Evaluating the Dose-Response of Sucralfate Binding to TDC:

Along with getting a sense of the reproducibility of the assay above, insights into the dose response relationship for Sucralfate binding to TDC was also achieved. The assay was performed using a fixed TDC concentration (2.5 mM) and pH (1.5) based on the results of the optimization experiments above and was evaluated for Sucralfate concentrations ranging from about 2 to 50 mg/mL using one lot of RLD product (RLD_04). The SCFeq (defined as the concentration of Sucralfate at half-maximal binding of TDC to Sucralfate at equilibrium) was determined to be about 16.14 mg/mL and the results indicated that the concentrations chosen for Sucralfate provided sufficient dynamic range for TDC binding to be able to delineate differences in binding between the test and RLD product in the assessments to follow. TDC binding was between about 2 and 75% for the chosen concentration range of Sucralfate for RLD_04 and about 18 to 85% for RLD_96. The results indicate that the assay has the ability to discriminate the binding of Sucralfate to TDC for Sucralfate concentrations of up to at least about 50 mg/mL.

3. Evaluation of the Range of Sucralfate Binding to Bile Acids Using Multiple Lots of RLD:

Once the dose-response of Sucralfate binding to TDC was observed using one lot of RLD product, assessments with additional available lots of RLD was commenced to determine the SCFeq value for each lot. The SCFeq value of each lot was calculated, which ranged between about 7.08 and 16.14 mg/mL for the four RLD lots (FIG. 3). These evaluations allow for the determination of the lot-to-lot variability of the RLD product, which can be used as the tolerance limits for comparative evaluation of test product performance.

The results provide sufficient confidence in the assay reproducibility and in the selectivity of the assay to detect differences in formulations compared to the RLD product. Furthermore, the range in % of TDC binding obtained from the evaluation for multiple lots of RLD provides insights into the variability of the RLD binding and can serve as limits of tolerance for the test formulations. The assay was deemed eligible and sufficiently qualified at this stage.

The table 2 below provides a range for the SCFeq value for each lot of RLD based on a 90% Confidence Interval:

RLD Lot # SCFeq 90% CI RLD_00 14.55 12.25 to 16.84 RLD_04 16.14 13.79 to 18.48 RLD_16 14.37 11.44 to 17.29 RLD_96 7.08 5.34 to 8.82

RLD 00, RLD_04, RLD_16 and RLD_96 are different lots of Carafate suspensions purchased from Forest Labs.

TABLE 3 Qualitative (Q1) and Quantitative (Q2) comparison of RLD and Test products for Sucralfate oral suspension Test formulations Lot #: RLD (CARAFATE ®) Test_50 Test_66 Lot #: RLD_45 Qty Qty Qty Ingredient Function (g/100 mL) (g/100 mL) (g/100 mL) Sucralfate API 10.00  10.00 10.00 Microcrystalline Cellulose NF Suspending Agent 1.00* 1.50 1.50 Colloidal Silicon dioxide NF Suspending Agent 0.20* 0.20 0.20 Methyl Cellulose USP Suspending Agent 0.25* 0.25 0.25 Glycerin 99.5% USP Bodying Agent 9.90^($ ) 9.90 9.90 Simethicone 30% Emulsion Antifoaming Agent 0.30* 0.30 0.30 Methyl Paraben Preservative 0.20* 0.20 0.20 Sorbitol 70% Solution USP Bodying Agent 27.0^($)     20.0 29.0 Water DI Filt. Vehicle Q.S. Q.S. to 100 mL Q.S. to 100 mL FD&C Red 40 Colorant Not listed in 0.66 0.657 patents/Literature Cherry Maraschino Artificial For Taste Not listed in 0.10 0.10 Flavor #2359 patents/Literature

Step 3: Assay Performance:

1. Evaluation of Binding of Sucralfate to TDC for the Initial Prototype Test Formulation (Test_50) and Comparison with that of the Reference Formulation(s):

Once the range of % binding and SCFeq values was determined using multiple RLD lots, the assay was performed using the early prototype test formulation (Test_50) using the same assay conditions (FIG. 4). The test formulation used here for comparison with the four RLD product lots is the initial prototype formulation that had undergone limited Q3 (microstructure; structural) characterization. In addition, the RLD characterization was also ongoing along with the determination of the CPPs (Critical Process Parameters) for the formulation manufacturing process. A SCFeq value of 20.06 mg/mL was obtained, higher than the SCFeq values obtained for the RLD product lots (7.08-16.14 mg/mL; RLD SCFeq Value range based on 90% CI interval: 5.34 to 18.48). This clearly demonstrates the ability of the assay to discriminate a “different” formulation and also corroborate the comparative findings of the physicochemical parameters evaluated to establish similarity in the formulation function. As the assay and the approach further develop, statistical analysis for establishing acceptance criteria for product similarity will be performed based on the factors evaluated in the assay validation stage. As this adds another bioassay measurement supportive of the postulated modes of action and the formulation CQAs (Critical Quality Attributes), the use of the in-vitro approach is further justified.

2. Evaluation of Binding of Sucralfate to TDC Using the Further Optimized Test Formulation (Test_66) and Comparison with that of the Reference Formulation(s):

As the formulation development efforts progressed, a further optimized test formulation (Test_66) was evaluated in for TDC binding to Sucralfate using the optimized assay conditions (FIG. 5). The SCFeq value obtained for Test_66 was 5.17 mg/mL, closer to the range of SCFeq values determined for the four RLD lots (7.08 to 16.14 mg/mL; RLD SCFeq value range based on 90% CI interval: 5.34 to 18.48) as compared to Test 50 (SCFeq=20.06 mg/mL) (FIG. 6). This demonstrates the ability of the assay to detect differences in the formulation composition (Q2 (quantitative) is different for Test_66 as compared to Test_50) and the manufacturing process between Test 66 and Test_50 products and will be used to guide further formulation development efforts.

Example 2

Cytoprotective Ability

Increase in the Potential Difference (Electrical Resistance) of Cells:

Optimized assay conditions are as follows: Indomethacin concentration was at 2 mM; pH for apical dosing solution was at 6.0. Sucralfate concentration was at 2.5 and 5 mg/mL. The assay duration was at 2 hours.

An ulcer develops as a result of an imbalance between aggressive factors and mucosal defensive mechanisms. The unstirred layer covering the mucosal luminal surface maintains a neutral microenvironment at the surface epithelial cells and is the first line of mucosal defense. The second line of mucosal defense is formed by a continuous monolayer of epithelial cells which secrete mucus and bicarbonate and generate prostaglandins. The plasma membrane lipid bilayer of the epithelial cells is hydrophobic, repelling acid- and water-soluble damaging agents. Interconnected by tights junctions, the mucosal epithelial cells form a barrier preventing back-diffusion of acid and pepsin.

Direct cellular damage due to necrotizing agents and the development of gastric lesions has been previously correlated with a decrease in the transmucosal electrical potential difference (PD) of the membrane barrier accompanied by net flux of Na⁺, Cl⁻, and H⁺ ions across the gastrointestinal mucosa [24]. A human intestinal epithelial cell culture system (Caco-2) exhibits morphological, biochemical, and functional characteristics similar to those of human intestinal mucosa in vivo, such as the expression of cell polarity and tight junctions. Caco-2 cells have been extensively used to study the barrier properties of human intestinal epithelium with regard to drug transport, paracellular flux of hydrophilic solutes, and transcellular passive diffusion of lipophilic solutes among many other applications [24]. PD (measured as the cell monolayer's transepithelial electrical resistance (TEER)), a measure of the integrity of the cell monolayer and its barrier properties, has also been studied and reported previously in evaluating the damage caused by acid and the protective effect of Sucralfate on the gastric mucosal barrier [24]. For all of the above reasons, the Caco-2 cell monolayer model was selected for evaluating the cytoprotective activity of Sucralfate and experiments were executed as per the assay eligibility determination process below. The results of each step are discussed below.

Step 1: Assay Development

-   -   1. pH optimization     -   2. Indomethacin concentration optimization     -   3. Assay duration optimization

Step 2: Assay Qualification:

-   -   1. Evaluate reproducibility     -   2. Evaluate assay selectivity by evaluating dose discriminating         ability of the assay     -   3. Establish the range in % TEER remaining using multiple lots         of RLD

Step 3: Assay Performance:

-   -   1. Evaluate the cytoprotective ability of Sucralfate using the         initial prototype formulation (Test_50) and compare with the RLD         product lot     -   2. Evaluate the cytoprotective ability of Sucralfate using the         further optimized test formulation (Test_66) and compare with         the RLD product lot

Results and Discussion:

Step 1: Assay Development:

1. Evaluation of Optimal pH Condition:

Considering the fact that the Caco-2 cell model mimics the human intestinal mucosa, and that the intraluminal pH of the small intestine ranges from approximately 6.0 to 7.4, evaluation of the damage induced by indomethacin (the chosen ulcerogenic agent, the use of which was previously reported [24]) was performed at these two pH conditions. The choice of 0.75 mM indomethacin was based on earlier observations reported for a similar evaluation using Caco-2 cells for this initial assay development stage [24]. For each pH condition, the damage was measured by comparing the TEER values of indomethacin-treated cell monolayers with those of control cell monolayers before and after the assay, and the % of TEER remaining (compared to the control treatment) was calculated. The results are illustrated below in FIG. 7. The results indicate that despite the long duration, at pH 7.4, a significant loss of TEER was not observed in the treatment with indomethacin. In contrast, for the assay with the pH condition of 6.0, approximately 50% of the TEER was lost by two hours and nearly 80% of the TEER was lost by the end of the assay duration of 5 hours. To demonstrate Sucralfate's ability to protect the cells from damage from necrotizing agents, it is desirable to induce sufficient damage to provide adequate dynamic range to demonstrate the cytoprotective ability of Sucralfate with sufficient sensitivity to discriminate between various formulations. Therefore, pH 6.0 was selected as the optimal pH condition on the apical (modeling the luminal) side for this assay. It was also noted that the majority of the damage was caused between 2 and 3 hours of duration; any additional damage after three hours was marginal. For this reason, further evaluations were performed at a truncated duration of 2 to 3 hours. See FIG. 7.

2. Optimization of Indomethacin Concentration:

Considering the fact that significant damage to the cells was induced post two hours in the assessment above and the fact that a shorter assay duration would be desirable for the purposes of experimental logistics, different concentrations (1, 1.5 and 2 mM) of indomethacin were tested to understand the extent of damage caused to the cell monolayers as a function of indomethacin concentration using apical pH of 6.0 and a 3 hour duration. Maximum damage was observed at 2 mM indomethacin as illustrated in FIG. 8. Therefore, a concentration of 2 mM was selected as the optimal concentration for this assay.

3. Optimization of Assay Duration:

Additional assessments were performed to ensure that sufficient damage was elicited by indomethacin at 2 mM using a 2-hour assay duration with and without Sucralfate at two different concentrations. The results as illustrated in FIG. 9, indicated that both the durations (2 and 3 hours) elicited sufficient damage, however, it appeared that the recovery of TEER for both concentrations of Sucralfate (1 and 5 mg/mL) was higher for the 2 hour assay duration as compared to the 3 hour assay duration. Therefore, 2 hours was chosen as the assay duration for further evaluations.

Based on these assessments, the above-listed assay conditions were chosen to qualify the assay by evaluating the assay reproducibility and the dose-dependent cytoprotective ability of Sucralfate.

Step 2: Assay Qualification:

1. Evaluation of Assay Reproducibility:

The reproducibility of the assay was evaluated by repeating the assay multiple times and calculating the % of TEER remaining after incubation with two concentrations of Sucralfate (2.5 and 5 mg/mL) using one lot of RLD product (RLD_04), as illustrated in FIGS. 10 and 11. It was observed that the assay was reproducible and the % TEER remaining (as compared to the control treatment) after 2 hours for the treatment co-dosed with Sucralfate ranged between about 81-104% for the 5 mg/mL treatment and about 65 to 106% for the 2.5 mg/mL treatment. Further assessments were then commenced to evaluate the assay selectivity.

2. Evaluation of the Assay Selectivity by Evaluating its Dose-Discriminating Ability:

Once the assay reproducibility was confirmed, further assessments were made to evaluate the dose-response of Sucralfate using multiple concentrations of Sucralfate RLD product lot (RLD_04). FIG. 12 below illustrates the results. An increase in cytoprotectiveness, as measured by the % of TEER remaining when compared to the control treatment, was seen with increasing Sucralfate concentrations up to 5 mg/mL. At 7.5 mg/mL, no additional increase in % TEER remaining was observed as compared to the 5 mg/mL treatment. The assay's dose-discriminating ability is evident only up to 5 mg/mL; therefore, further assessments will be performed at concentrations of 5 mg/mL or less.

3. Establishing the Range of % TEER Remaining Using Multiple Lots of RLD:

Four lots of RLD product were then evaluated for cytoprotective ability to evaluate the range of % TEER remaining using two concentrations of Sucralfate (2.5 and 5.0 mg/mL). This would allow the establishment of tolerance limits for comparison with the Test product lots. For the 2.5 mg/mL concentration, the range for % of TEER remaining for all four lots of RLD product was observed to be 23.3 to 74.8% as illustrated in FIG. 13 and for the 5.0 mg/mL concentration, the range of % TEER remaining for all four lots of RLD product was observed to be 64.2 to 92.5% as illustrated in FIG. 14.

Step 3: Assay Performance:

1. Evaluation of the Cytoprotective Ability of Sucralfate for the Initial Prototype Formulation (Test_50) and Further Optimized Formulation (Test_66) and Comparison with the RLD Product Lots:

The initial prototype test formulation lot (Test_50) and the further optimized test formulation lot (Test_66) were then evaluated and compared with a RLD product lot (RLD_04) using two concentrations of Sucralfate (2.5 and 5 mg/mL). The results exhibited cytoprotective effects for both RLD and test product lots as illustrated in FIG. 15 for both concentrations tested. For the RLD product lot (RLD_04), the % TEER remaining at the 2.5 mg/mL concentration was found to be about 63.6% and the % TEER remaining for the 5 mg/mL concentration was found to be about 90.3%. For the initial prototype formulation (Test_50), using the 2.5 mg/mL treatment, the % TEER remaining was about 77.3%, which was not within the range of RLD performance in this assay for this concentration (23.3% to 74.8%). Moreover, a dose-response was not observed for this Test lot (Test_50) and both the 2.5 and 5.0 mg/mL treatment groups had a similar % TEER remaining (2.5 mg/mL: 77.3% and 5.0 mg/mL: 78.8%) after the two hour incubation duration. For the more optimized test formulation (Test_66), the % TEER remaining for the 2.5 mg/mL treatment was 63.3%, which was within the RLD range of performance and a dose-response was observed for the two concentrations of Sucralfate (2.5 mg/mL: 63.3% and 5.0 mg/mL: 85.0%), similar to that observed for the RLD lot (RLD_04).

These findings again confirm the reproducible and selective nature of the assay. In addition, it demonstrates the ability of the assay to distinguish between formulations that are different as compared to the RLD product by clearly demonstrating a lack of dose-response for the Test_50 formulation, a formulation that was prepared in the very early stages of formulation development without having completed the reverse engineering and characterization of the RLD. Test_66 was prepared subsequently, after the reverse engineering of the RLD was completed along with the physicochemical characterization of the RLD and delineation of some of the CPPs (Critical Process Parameters). The results for the % TEER remaining for Test_66 not only demonstrated the expected dose-dependent response, but they were also “similar” to that of the RLD lot. This also indicates that this assay along with the remaining proposed assays can be used during the formulation optimization stages to prepare a final formulation that is not only close to the RLD in terms of function (as identified by the physicochemical characterization), but also close to the RLD in terms of the outcomes of the biological assays based on the postulated modes of action.

In summary, the Caco-2 cell monolayer model is a well-established, widely used, and accepted model that can be used as a tool to evaluate one of the key mechanisms of action of Sucralfate. The model is also discriminatory of formulation differences and can be used for formulation optimization followed by pivotal equivalence assessments. The results from this study can build sufficient weight of evidence to demonstrate similarity RLD and test product and to establish therapeutic equivalence between the test product and the RLD product. See FIGS. 16-18.

DISCUSSION

BE (bioequivalence) assessment is essential for approval of generic drug products. The requirements for a BE study vary greatly from product to product for various reasons. For some products, such as Sucralfate oral suspension, these requirements can be a huge barrier to market entry for generic products despite the need for the product and a lack of availability of any interchangeable product. The underlying scientific basis for the BE assessment for the generic drug product is that it should be safe and as effective as the reference drug product.

For the majority of drug products, the requirements of a BE study are both ethical and scientifically appropriate in accurately deducing a product's therapeutic equivalence. In the case of Sucralfate oral suspension, as of today there is no guidance from the FDA for assessing BE for Sucralfate. The draft guidance on Sucralfate, which was published in July 2014 by the FDA, required a three-arm, placebo-controlled, in-vivo clinical endpoint study in healthy males and females with dyspepsia symptoms and active duodenal ulcer disease, verified at screening endoscopy. Additionally, the enrolled patients had to be H. pylori negative or continue to have the presence of an ulcer after appropriate treatment and eradication of H. pylori. Furthermore, the duration of this study was 8 weeks, and not only is it challenging to recruit subjects for the study but it is also unethical to administer a placebo treatment to patients and allow for their continued suffering. The FDA acknowledged the challenge of this BE study requirement and encouraged sponsors to submit proposals for in vitro studies or any other methods for evaluating BE for this drug product in the 2014 Guidance.

For Sucralfate, the site of action is local in the GI tract. The drug product dissolution and transit controls the presentation of drug to the site of action and the drug's plasma concentration is downstream from the site of action and irrelevant to the clinical effect. It also has very low systemic availability and is not detected in plasma. For other drugs that are absorbed sparingly or not at all and intended for local activity, such as cholestyramine and sevelamer, in-vitro binding studies have been recommended by the US FDA to demonstrate BE and are deemed sufficient based on the mechanism of action of these drugs.

The mechanism of action of Sucralfate is non-systemic and, in general, Sucralfate exerts its effects by protecting the gastric mucosa against various irritants and providing a cytoprotective effect by enhancing natural mucosal defense mechanisms. Because various mechanisms of action have been postulated for Sucralfate, in-vitro binding studies, although critical in establishing BE, are not by themselves sufficient for establishing BE. For this reason, the proposed approach takes into consideration the interaction of Sucralfate throughout the upper GI tract where the drug product is presented, and the CQAs (Critical Quality Attributes) that determine formulation function are evaluated in conjunction with bioassays that support the postulated modes of action.

The approach includes similarity-by-design and begins with the development of a test product formulation which is qualitatively and quantitatively the same as the RLD with respect to the active and inactive ingredients. Based on the target product profile, the CQAs that affect the formulation function and biological effects have been identified and the critical material attributes and process parameters will be optimized until similarity of the test product in formulation function and in the bioassays with the RLD product is achieved. Once a similar prototype formulation is identified, scale-up batches will be prepared and evaluated for similarity using all assays that can be validated appropriately in preparation for an ANDA submission.

The purpose of the data generated thus far is to demonstrate the utility of each assay individually in evaluating the corresponding CQA that contributes to product efficacy and to demonstrate the use of equivalence agreement between orthogonal measurements to ensure and establish therapeutic equivalence. A clinical endpoint study has been acknowledged by the FDA and the scientific community to be the least accurate way to establish BE, and the use of multiple validated assays such as the ones described above can be a more accurate way to establish BE. An in-vitro based bioequivalence approach is accepted for several complex drug products such as acyclovir ointment, cyclosporine ophthalmic emulsion and tobradex ophthalmic emulsions based on sound scientific principles.

The formulation development scientists initiated several activities in parallel, including API (Active Pharmaceutical Ingredient) sourcing and evaluation, reverse engineering of the RLD formulation, review of chemistry, preparation of the target product profile, identification of the CMAs (Critical Material Attributes) and CPPs (Critical Process Parameters) that affect the CQAs (Critical Quality Attributes), equipment and methodologies required for various assessments, etc. Various R&D scale batches were prepared in the process and data was generated for selective parameters for each batch. Analytical techniques are well established for physicochemical/Q3 (microstructure/structural) characterization of oral suspensions with some techniques requiring equipment that is readily available at the in-house facility. As the R&D batches were still being prepared and further optimized, an early lot of the test formulation prototype (Test_50) was selected for evaluations in biological assays to understand and discern any differences in formulation performance that can be identified in these assays when compared to the RLD lots. For all of the biological assays performed for Test_50 it was evident that it was “different” as compared to the multiple lots of RLD.

Focusing on the binding assessments, the SCFeq for the bile acid binding was comparatively higher for Test_50 as compared to the RLD lots (about 20.06 vs. about 7.08-16.14 mg/mL; RLD SCFeq value range based on about 90% CI interval: about 5.34 to 18.48), which suggests stronger binding/affinity of Sucralfate from the RLD formulation as compared to the test formulation. It is to be noted that Sucralfate binding in general is highest at low pH conditions; however, due to the pKa of the bile acids, at lower pH the non-ionized form of the bile acid conjugates predominate and tend to precipitate. Regardless, similarity must be demonstrated in the bile acid binding of Sucralfate and the assay clearly has the ability to discriminate between different formulations.

For the cytoprotective assay using Caco-2 cell monolayers, the % TEER remaining with Test_50 was lower compared to the % TEER remained for the RLD lots (Test_50: 78.8% vs. RLD range: 82 to 85%), which indicated somewhat less recovery of cells from the damage induced by indomethacin as compared to the recovery seen using the RLD lots. These initial assessments demonstrate that the initial prototype formulation (Test_50) is “different” compared to the RLD lots, as one would expect considering that various aspects of formulation design were not optimized including the Q2 (quantitative) and the CPPs (Critical Process Parameters).

Similarly, although the results for Test_66 also indicate that it is a “different” formulation, the optimization efforts commenced by the formulation development team are evident in the results.

With regard to bile acid binding, the SCFeq value for Test_66 (about 5.17) was much closer to the RLD range (about 7.08 to 16.14; RLD SCFeq Value range based on about 90% CI interval: about 5.34 to 18.48) as compared to the Test_50 (about 20.06) formulation. As mentioned previously, the Q2 of Test_66 is different as compared to Test_50, along with differences in process parameters such as homogenization time and order of addition of simethicone. These optimization efforts have clearly resulted in a formulation closer to the RLD and the bioassay is able to distinguish the known formulation differences between the Test_50 and Test_66 formulations.

For the cytoprotective assay, Test_66 is closer to the RLD, with the % TEER remaining being 85% (vs. 64.2-92.5% remaining for RLD lots) at 5 mg/mL, whereas only 78.8% of the TEER is remaining for the Test_50 formulation treatment. In addition, a dose-response was not observed using the Test_50 formulation, which is expected based on all optimization and qualification evaluations with the RLD. The dose-response is observed with Test_66, as expected. This further reinforces the positive direction of the formulation optimization efforts and demonstrates that these assays can be valuable tools throughout formulation development and for establishing BE.

Example 3

Delay of TDC Migration Across Sucralfate Barrier

In addition to forming a barrier, sucralfate oral suspension reportedly acts by adsorbing bile salts, which may prevent the bile salts from disrupting the gastric mucosal barrier [28]. It is yet to be determined whether the clinical efficacy of sucralfate is a result of bile salt adsorption/depletion (by sucralfate that has not yet formed a barrier) or barrier formation by sucralfate that results in a delay of bile salt migration to the gastroduodenal mucosa and the mucosal defect [19]. The barrier formation is multifactorial and formulation-dependent, driven by various formulation-dependent factors such as the physicochemical properties of the formulation along with binding and adsorption properties of sucralfate [29]. Therefore, the similarity in the formation of this complex barrier, which results in protection from noxious substances such as bile salts, is a key characteristic to be evaluated using the RLD and test formulations to ensure equivalence in the test and RLD products' performance. The TDC diffusion assay measures the effect of test and RLD formulations on TDC diffusion and thus the ability of the formulations to hinder bile salt disruption of the mucosa. The assay should include an appropriate barrier, an analytical method for quantifying TDC, and an assessment of TDC diffusion by monitoring the change in TDC concentration over time.

The TDC diffusion (delay of TDC migration) assay along with the bile salt binding and the cytoprotective assays can be tailored to reflect specific physiological conditions of the environment of actual use. Equivalence in the above in vitro bioassays correlates with equivalence in in vivo endpoints, ensuring BE even where small differences between test and reference formulations exist (i.e., the formulations are not quantitatively the same).

1. Method Summary

The assay is used to determine sucralfate's ability to inhibit TDC diffusion using an in vitro diffusion system.

2. Equipment

Equipment includes: pH meter; Balance; Refrigerator; VWR Scientific Products Standard Mini Vortexer; Precision Scientific water bath with shaker; Calibrated thermometer; VWR Stirring plate; Calibrated Adjustable Micropipettes 5 μL, 10 μL, 20 μL, 100 μL, 200 μL, 1,000 μL, 2,000 μL, 5 mL, 10 mL; LC-MS/MS: Applied Biosystems API 4000 Mass Spectrometer with Waters Acquity UPLC; Vacuum filter; Plate mixer: Eppendorf and VWR; Centrifuge: Beckman GS-6R; and any additional general laboratory equipment.

3. Materials

Materials include: Sucralfate oral suspension (SCF), 100 mg/mL: Reference Listed Drug (RLD) or Test Product; Sucralfate Placebo; Acetic acid; Sodium acetate Milli-Q H₂O; Albumin from Human Serum (HSA); Transwell® Permeable Supports 12 mm Diameter Inserts; and Sodium Taurodeoxycholate Hydrate (TDC). A substitution of comparable grades of materials from different vendors is acceptable unless specifically stated.

4. Reagents

Reagents include: 2 N acetic acid and Acetate buffer pH 4.55% HSA in acetate buffer pH 4.5.

5. Dosing Solutions

Dosing solutions include: 200 mM TDC stock in the acetate buffer pH 4.5; TDC solution in 5% HSA in acetate buffer pH 4.5 (without any sucralfate formulation); and Dosing solution with Sucralfate.

TABLE 4 Example Treatment Groups Volume of Total Sucralfate Volume formulation of Group Treatment Concentration (μL) Replicates Dosing 1 Control 0.5 mM TDC 300 μL 3 3 mL Control (no acetate formulation) buffer pH 4.5 2 Sucralfate 0.5 mM TDC + 300 3 3 mL formulation 10 mg/mL Sucralfate Lot __ 3 Sucralfate 0.5 mM TDC + 600 3 3 mL formulation 20 mg/mL Sucralfate Lot __ 4 Sucralfate 0.5 mM TDC + 150 3 3 mL formulation  5 mg/mL Sucralfate Lot __

6. ASSAY Procedure

Assay procedure includes: Dosing the Trans-well inserts (n=3 per sucralfate treatment). Pipette 1.5 μL of acetate buffer pH 4.5 into the receiver chamber to “pre-wet” the insert for 5 minutes. Start the timer after addition to the first chamber. After completion of the 5 minutes, load 500 μL of dosing solution in the insert. Donor time-points: 120 min. Sampling volume: 50 μL. Receiver time-points: 15, 30, 60, 90 and 120 min. Sample volume: 200 μL. Measure all donor samples, receiver samples and the TDC dosing solution (control dosing solution) by LC-MS/MS. Dilute the samples as appropriate for sample preparation.

7. Data Processing

Calculate cell-free apparent permeability (P_(app)) in Excel and obtain a plot average P_(app) for each Sucralfate treatment group as a bar graph representation in GraphPad Prism.

Results

The objective of this TDC diffusion (delay of TDC migration) assay was to assess the in vitro diffusion (delay of migration) of taurodeoxycholate (TDC) in the presence of sucralfate suspension. In the FIG. 19, TEST 06 falls within the range of the Papp measurements for different lots of the Carafate suspension product, the reference listed drug (RLD_72, RLD_66, RLD_63, RLD_23, RLD_10 and RLD_02), indicating that TEST_06 with respect to the assay is equivalent to the RLDs. In contrast, the non-formulated aqueous suspension of sucralfate i.e., the active pharmaceutical ingredient (API), has a measurement significantly outside of the range of values for the various lots of the RLDs, indicating the importance of formulating the API for the delay in migration of TDC and its effectiveness as a barrier against noxious substances. Lastly, the delay of migration/diffusion assay was conducted with a representative bile salt, TDC; however, this delay in migration/diffusion assay may be conducted with bile acid and/or a conjugated bile acid or a combination thereof, or with other bile acid, bile salt and/or a conjugated bile acid or a combination thereof, as disclosed in herein. Furthermore, while we have disclosed a Corning Transwell®-based assay, assays using other apparatuses and analyses may be used to examine delay in migration/diffusion of bile acid, bile salt and/or a conjugated bile acid or a combination thereof, in the presence of sucralfate suspension product. It would be clear to one skilled in the art that other assays may be used in order to achieve the desire goal of assessing delay in migration or diffusion of bile acid, bile salt and/or a conjugated bile acid or a combination thereof, in the presence of sucralfate as supplied in a sucralfate suspension product.

Example 4

Bile-Acid Binding Assessment Using Taurodeoxycholate (TDC)

Objective

To compare the adsorption of sodium taurodeoxycholate (TDC) to sucralfate formulations

Experimental Conditions

Equipment

Equipment includes: pH meter; Incubator shaker; Beckman Coulter Microfuge™ 18 centrifuge; Analytical Balance; Calibrated Adjustable Micropipettes 100 μl, 200 μl, 1,000 μl, 2,000 μl, 5,000 μl, 10,000 μl; Vortexer; and any additional general laboratory equipment.

Materials

Materials include: Sodium taurodeoxycholate; Sucralfate oral suspension (SCF), 100 mg/mL: Reference Listed Drug (RLD) or Test Product; Potassium phosphate monobasic; Sodium hydroxide (ION); Hydrochloric acid (HCl), 37% concentration; Eppendorf 2.0 mL centrifuge tubes; 4-mL amber vials, and other sizes as needed; 96-Well Polypropylene Plates (2.0 mL); and Simulated Intestinal Fluid (SIF) Without Enzymes.

Assay Procedure

Pre-warm SIF without enzyme at pH 6.0 and confirm pH (adjust, if necessary) prior to beginning the assay. Obtain 6 mL of sucralfate formulation (reference or test/exhibit batch) and dilute 1:1 with 6 mL of 0.1 N HCl and incubate for one hour. Mix 250 μL of acid pre-treated SCF with 250 μL of TDC solutions at varying concentrations in 2-mL round bottom tubes. Incubate all tubes in an incubator shaker set to 37° C. while shaking. Centrifuge all tubes at 10,000 RPM for 10 minutes. Collect 200 μL of supernatant (being careful not to disturb the pellet) into a 96-well plate for analysis. Store all samples at 4° C. until ready to be analyzed on LC-MS/MS.

Data Treatment and Analysis

Langmuir Constants

The amount of TDC (adsorbate) adsorbed on sucralfate (adsorbent) is calculated from the difference between initial TDC concentration and the concentration in the supernatant at the end of incubation. The adsorption of adsorbate onto adsorbent surface at constant temperature can be described by Langmuir equation (1) or upon rearranging by equation (2)

x/m=k ₁ *k ₂ *C _(eq)/(1+k ₁ *C _(eq))  (1)

C _(eq)/(x/m)=1/(k ₁ *k ₂)+C _(eq) /k ₂  (2)

Where:

C_(eq) (mM)=concentration of TDC remaining in the supernatant after incubation

x/m (mmol/g)=amount of TDC (mmol) bound per the amount of sucralfate (g)

k₁ (1/mM or L/mmol)=affinity constant

k₂ (mmol/g)=Langmuir capacity constant

A plot of C_(eq)/x/m versus C_(eq) yields a straight line with a slope (a) and intercept (b) after applying regression analysis. The affinity constant k₁ (1/mM or L/mmol) and Langmuir capacity constant k₂ (mmol/g) are calculated from the intercept and the slope

k ₁ =a/b  (3)

k ₂=1/a  (4)

Results

The objective of this TDC binding assay was to evaluate and compare the adsorption of TDC to sucralfate oral suspension. In the FIG. 20, TEST_06 and Test_43 falls within the range of the k₂ values for the two lots of RLD. Carafate suspension product RLD_02 and RLD_72, indicating that TEST_06 and Test_43 with respect to the assay is equivalent to the RLD.

Additionally, Test_06 appears to be the only Test formulation which is deemed to be equivalent in all three assays (FIGS. 18, 19 and 20) and therefore, the conclusion of equivalence for this Test_06 formulation in all three assays can be deemed reliable.

REFERENCES

-   1. Carafate Suspension Product PIL. -   2. McCullough, R. W., IBS, NERD and functional dyspepsia are     immuno-neuronal disorders of mucosal cytokine imbalances clinically     reversible with high potency Sucralfate. Medical hypotheses, March     2013, Volume 80, Issue 3, Pages 230-233. -   3. FDA response to Consultation Re: Biocraft Sucralfate Tablet     Submission on Apr. 27, 1988. -   4. Draft Guidance on Sucralfate. Recommended July 2014, FDA. -   5. http://www.fda.gov/ohrms/dockets/ac/05/briefing/2005-137B1_07     Nomenclature.pdf. -   6. Code of Federal regulations. Title 21, Volume 5; CITE:     21CFR320.24. -   7. The Merck Index. 10th ed. Rahway, N.J.: Merck Co., Inc.,     1983., p. 1273. -   8.     http://intranet.tdmu.edu.te.ua/data/kafedra/internal/pharmakologia/classes_stud/en/nurse/and/ptn/Pharmacology/2r/15%20Drugs     %20Affecting %20Gastrointestinal%20System %20and %20Nutrition.html. -   9. http://omedicine.info/en/Sucralfate.html. -   10. http://www.ncbi.nlm.nih.gov/pubmed/8370300. -   11. F. Halter, Daniel Hollander M. D., Guido N. J., Tytgat M. D.     Sucralfate From Basic Science to the Bedside. -   12. Patent on Sucralfate suspension.     (http://www.google.co.in/patents/U.S. Pat. No. 5,563,258). -   13. Cutting, K. F., Wound exudate: composition and functions.     British Journal of community nursing. February 2003. Pages 4-9. -   14. Ito, Y., Onada, Y., Nakamura, S. Tagawa, K., Fukushima, T.,     Suguwara, Y., and Takaiti, O. Effects of new anti-ulcer drug ecabet     sodium (TA-2711) on pepsin activity. II. Interaction with substrate     protein, Japan J. Pharmacol., 62, 175-181; 1999 -   15. Kawakami, K., Yasuda, M., Ishii, K., Kokusenya, Y., and Sato, T.     A kinetic study of protein binding to ecabet sodium using     quartz-crustal microbalance Chem. Pharm. Bull. 47(7)919-922; 1999. -   16. Methods Find Exp Clin Pharmacol. 1989; 11 Suppl 1:19-25. The     gastric mucosal barrier. Clamp JR1, Ene D. -   17. Reshetnyak, V. Physiological and molecular biochemical     mechanisms of bile formation. World J. of gastroenterol. 2013 Nov.     14; 19 (42): 7341-7360. -   18. Duane, W. and Wiegand, D. Mechanism by which bile salt disrupts     the gastric mucosal barrier in the dog. The Journal of clinical     investigation, Volume 66, November 1980 1044-1049. -   19. Graham, D. Y., Sackman, J. W., Giesing, D. H., and Runser, D. J.     In Vitro Adsorption of Bile Salts and Aspirin to Sucralfate.     Digestive diseases and sciences, Vol. 29, No. 5 (May 1984), pp.     402-406. -   20. Furukawa, O., Matsui, H., & Suzuki, N. (1997). Effects of     Sucralfate and its components on acid- and pepsin-induced damage to     rat gastric epithelial cells. Japanese Journal of Pharmacology, 75:     21-25. -   21. Sigma-Aldrich (n.d.). Technical bulletin for Protease     Colorimetric Detection Kit (Product Code PC0100). Retrieved from     http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/pc0100bul.pdf. -   22. Okabe, S., and Amagase, K. An overview of acetic acid ulcer     models—The history and state of the art of peptic ulcer research.     Biol. Pharm. Bull. 28(8) 1321-1341.2005. -   23. Dobrozsi, D. J., Smith, R. L., Sakr, A. A. Comparative     mucoretention of Sucralfate suspensions in an everted rat esophagus     model. International Journal of Pharmaceutics 189. 81-89. 1999. -   24. Tang, A. S., Chikhale, P. J., Shah, P., Borchardt, R. (1993)     Utilization of a human intestinal epithelial cell culture system     (Caco-2) for evaluating cytoprotective agents. Pharmaceutical     Research 10 (11): 1620-1626. -   25. Waiver of In vivo Bioavailability and bioequivalence studies for     Immediate-release solid oral dosage forms based on a     biopharmaceutics classification system. Guidance for industry. May     2015, FDA. -   26. FDA definition of a suspension:     https://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4241B1-02-30-FDA-Topical%20Dosage%20Forms%20Definitions%2001d%20%20.pdf -   27. SCF binding to free bile acids:     https://www.ncbi.nlm.nih.gov/pubmed/2682004. -   28. Nagashima, R. (1981) Development and characteristics of     Sucralfate. Journal of Clinical Gastroenterology 3 (suppl 2):     103-110. -   29. Bighley L D, Giesing D. Sucralfate. A new concept in ulcer     therapy. In: Peptic ulcer disease: an update. New York: Biomedical     Information, 1979:307-20. 

1.-5. (canceled)
 6. A sucralfate suspension sample so obtained by a method for determining in vitro bioequivalence of a sucralfate suspension sample to a sucralfate suspension RLD comprising: a) contacting the sucralfate suspension sample with a bile acid, bile salt and/or a conjugated bile acid or a combination thereof, so as to permit sucralfate-bile interaction, quantifying the interaction so as to obtain quantified values of the interaction and comparing said values to reference values for the sucralfate suspension RLD; and b) contacting the sucralfate suspension sample with a cell culture system in the presence of a Non-Steroidal Anti-Inflammatory Drug (NSAID) or a nonselective inhibitor of cyclooxygenase (COX) 1 and 2 so as to inhibit cell damage induced by the NSAID or nonselective inhibitor of COX 1 and 2 in the cell culture system, quantifying the cell damage so as to obtain quantified value(s) for the sucralfate suspension sample and comparing the said value(s) to reference values for the sucralfate suspension RLD; wherein the reference values for the sucralfate suspension RLD in (a) are derived from a set of values obtained from quantifying the sucralfate-bile interaction for 2 or more lots of the sucralfate suspension RLD; wherein sucralfate-bile interaction is interaction between sucralfate and bile acid, bile salt and/or conjugated bile acid or a combination thereof; wherein the reference values of the sucralfate suspension RLD in (b) are derived from a set of values obtained from quantifying the reduction in cell damage for 2 or more lots of the sucralfate suspension RLD; and wherein the sucralfate suspension sample is to have said in vitro bioequivalence to a sucralfate suspension RLD when the values determined in (a) and (b) for the sucralfate suspension sample are within the reference values in (a) and (b) for the sucralfate suspension RLD, respectively.
 7. The sucralfate suspension sample of claim 6, wherein the conjugated bile acid (a) is selected from the group consisting of glycocholate, glycochenodeoxycholate, taurochenodeoxycholate and taurodeoxycholate or a combination thereof.
 8. The sucralfate suspension sample of claim 6, wherein the bile acid is or comprises a taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7).
 9. The sucralfate suspension sample of claim 6, wherein the bile salt is or comprises a sodium taurodeoxycholate (CAS No.: 1180-95-6).
 10. The sucralfate suspension sample of claim 6, wherein the conjugated bile acid is or comprises taurodeoxycholate.
 11. The sucralfate suspension sample of claim 6, wherein the bile salt comprises of a bile acid and a cation selected from the group consisting of an alkali metal, alkaline earth metal, transition metal, ammonium, amine and a quaternary ammonium.
 12. The sucralfate suspension sample of claim 11, wherein the alkali metal is selected from the group consisting of lithium, sodium, potassium, rubidium and cesium.
 13. The sucralfate suspension sample of claim 11, wherein the alkaline earth metal is selected from the group consisting of beryllium, magnesium, calcium, strontium and barium.
 14. The sucralfate suspension sample of claim 11, wherein the transition metal is selected from the group consisting of chromium, molybdenum, manganese, iron, cobalt, nickel, copper, silver, gold, zinc and cadmium.
 15. The sucralfate suspension sample of claim 11, wherein the amine is selected from the group consisting of methylamine, methoxylamine, methylenediamine, bromoethylamine, chloroethylamine, fluoroethylamine, dimethylamine, ethylenediamine, diethylamine, cystamine, aniline, nitro-benzenediamine, phenylenediamine, tris(2-chloroethyl)amine, tromethamine, ethanolamine, diethanolamine and triethanolamine.
 16. The sucralfate suspension sample of claim 11, wherein the quaternary ammonium is selected from the group consisting of tetramethylammonium, tetrabutylammonium, tetraethylammonium, triethylmethylammonium and tributylmethylammonium.
 17. The sucralfate suspension sample of claim 6, wherein the bile salt is sodium or potassium salt of a bile acid.
 18. The sucralfate suspension sample of claim 6, wherein the sucralfate-bile interaction is binding of bile acid, bile salt or conjugated bile acid or a combination thereof by sucralfate.
 19. The sucralfate suspension sample of claim 18, wherein the binding is in vitro equilibrium binding.
 20. The sucralfate suspension sample of claim 18, wherein binding is in vitro kinetic binding.
 21. The sucralfate suspension sample of claim 18, wherein the binding is adsorption of bile acid, bile salt or conjugated bile acid or a combination thereof to sucralfate.
 22. The sucralfate suspension sample of claim 18, wherein the interaction or association delays the migration of bile acid, bile salt or conjugated bile acid or a combination thereof in the presence of sucralfate. 23.-112. (canceled)
 113. The sucralfate suspension sample of claim 6, wherein the bile acid is selected from the group consisting of allocholic acid (3alpha,7alpha,12alpha-trihydroxy-5alpha-cholanoic acid; CAS: 2464-18-8), 5alpha-deoxycholic acid (3alpha,12alpha-dihydroxy-5alpha-cholan-24-oic acid), bitocholic acid, chenodeoxycholic acid (3alpha,7alpha-dihydroxy-5beta-cholan-24-oic acid; CAS: 474-25-9), cholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 81-25-4), deoxycholic acid (3alpha,12alpha-dihydroxy-5beta-cholanic acid; CAS: 83-44-3), glycochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]acetic acid; CAS No.: 640-79-9), glycocholic acid (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oylglycine; CAS: 475-31-0), hyocholic acid (3alpha,6alpha,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 547-75-1), hyodeoxycholic acid (3α,6α-Dihydroxy-5β-cholan-24-oic acid; CAS: 83-49-8), isochenodeoxycholic acid (3beta,7alpha-dihydroxy-5beta-cholanic acid; CAS: 566-24-5), 3beta,12alpha-Dihydroxy-5beta-cholanoic acid (CAS: 570-63-8), isolithocholic acid (3beta-Hydroxy-5beta-cholan-24-oic acid; CAS: 1534-35-6), isoursodeoxycholic acid (3beta,7beta-dihydroxy-5beta-cholan-24-oic acid; CAS: 78919-26-3), 12-epideoxycholic acid, lithocholic acid (3alpha-hydroxy-5beta-cholanic acid; CAS: 434-13-9), alpha-muricholic acid (3alpha,6beta,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 2393-58-0), beta-muricholic acid (3alpha,6beta,7beta-trihydroxy-5beta-cholan-24-oic acid; CAS: 2393-59-1), omega-muricholic acid, murideoxycholic acid (3alpha,6beta-dihydroxy-5beta-cholanic acid), beta-phocaecholic acid (CAS: 105369-89-9), taurochenodeoxycholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CAS No.: 516-35-8), taurocholic acid (2-[[(4R)-4-[(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]ethanesulfonic acid; CAS No.: 81-24-3), taurodeoxycholic acid (2-(((3alpha,5beta,12alpha)-3,12-dihydroxy-24-oxocholan-24-yl)amino)-ethanesulfonic acid; CAS No.: 516-50-7), ursocholic acid (3alpha,7beta,12alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 2955-27-3), ursodeoxycholic acid (3alpha,7beta-dihydroxy-5beta-cholan-24-oic acid; CAS: 128-13-2), and vulpecholic acid (1alpha,3alpha,7alpha-trihydroxy-5beta-cholan-24-oic acid; CAS: 107368-95-6) or a combination thereof.
 114. The sucralfate suspension sample of claim 6, wherein the bile salt is salt of a bile acid.
 115. The sucralfate suspension sample of claim 6, wherein the conjugated bile acid (a) is the taurine or glycine conjugate of a bile acid. 